Summary The long-range goal of this proposal is to obtain a crystal structure(s) of a new conformation of the lactose permease of Escherichia coli (LacY) in order to understand the mechanism of lactose/H+ symport. LacY is a paradigm for a huge group of structurally related membrane transport proteins (the Major Facilitator Superfamily), as well as for membrane proteins in general. Our first X-ray crystal structure of a conformationally crippled mutant of LacY (C154G) represents a major breakthrough as the first structure of a cation-coupled symporter. In the past grant period, we also solved an X-ray structure of wild-type LacY to a resolution of 3.6 [unreadable], which represents another highly significant breakthrough, as the accomplishment took well over a decade and required development of a new approach that has general applicability. In addition, we improved the resolution of the C154G LacY structure to a resolution of 2.95 [unreadable]. However, all available structures from the wild type and the conformationally crippled mutant display the same global fold. All structures are composed of symmetrical N- and C-terminal domains, each with 6 transmembrane &#945;-helices most of which are irregular. There is a large internal hydrophilic cavity open to the cytoplasmic side, which clearly represents an inward-facing conformation, as the periplasmic side is tightly closed. The residues that play major roles in galactopyranoside recognition and H+ translocation are clustered near the apex of the cavity and are inaccessible from the periplasmic side. A mechanism consistent with the structure and various biochemical and biophysical approaches has been proposed, the heart of which is alternative accessibility of the sugar-binding site to either side of the membrane. Therefore, it is critical to obtain structures in a different conformations. We have obtained diffracting crystals of mutants and constructs that are approaching a resolution suitable for structure determination. The main aims of the proposal are (i) to obtain structures of other conformations of LacY;(ii) to obtain a structure of LacY that diffracts to a resolution sufficient to visualize bound water. We will combine mutagenesis and chemical modification to induce conformations different from the inward-facing confirmation. The proposed structures will be invaluable for understanding the mechanism of cation-coupled membrane transporters, a class of proteins that plays essential roles in many cellular functions and has broad impact on biology and medicine.